1. Field of the Invention
This invention relates to a method of producing L-phenylalanine by treating an N-acylphenylalanine alkyl ester in L form with an enzyme in the presence of water.
2. Description of the Related Art
Various methods are so far known for producing L-phenylalanine by treatment of an L-phenylalanine precursor with an enzyme. For instance, for producing L-phenylalanine by treating an L-phenylalanine precursor with an hydrolyzing enzyme, there are known, among others, a method which comprises treating an N-acyl-DL-phenylalanine with aminoacylase to thereby selectively hydrolyze an N-acyl-L-phenylalanine alone for conversion thereof to L-phenylalanine [cf. U.S. Pat. No. 3,386,888 and Bull. Agr. Chem. Soc. Japan, 21, 300 (1957)], a method which comprises treating a DL-phenylalanine alkyl ester with esterase to selectively hydrolyze an L-phenylalanine alkyl ester alone for conversion to L-phenylalanine [cf. J. Biol. Chem., 203, 755 (1953)], and a method which comprises hydrolyzing 75-benzylhydantoin by treatment with hydantoinase for its conversion to L-phenylalanine (cf. Japanese Patent Publication No. 2274/79). Also known as an alternative is a method which comprises treating an N-acyl-DL-phenylalanine methyl ester with serine proteinase or esterase to thereby selectively hydrolyze an N-acyl-L-phenylalanine methyl ester alone to give the corresponding N-acyl-L-phenylalanine, separating the latter from the unreacted N-acyl-D-phenylalanine methyl ester and hydrolyzing the same with hydrochloric acid for conversion thereof to L-phenylalanine [cf. U.S. Pat. No. 4,262,092 and Synthesis, 1041 (1983)].
Hydrolyzing enzymes, such as aminoacylase and esterase, in many cases exhibit high hydrolyzing activity under nearly neutral conditions. Therefore, for treating acidic or basic L-phenylalanine precursors such as an N-acyl-DL-phenylalanine or a DL-phenylalanine alkyl ester with such hydrolyzing enzymes, it is necessary to adjust the pH of a starting material-containing solution to a value near neutrality in advance by adding a base or an acid to said solution. The reaction mixture obtained by such hydrolysis contains, in addition to L-phenylalanine, which is the desired product, an unreacted N-acyl-D-phenylalanine or an unreacted D-phenylalanine alkyl ester, which occurs as a salt with the above-mentioned base or acid. Generally, L-phenylalanine crystallizes out from such reaction mixture. In that case, also the above salt is ready to crystallize out, so that, for recovering L-phenylalanine in a satisfactorily high purity, a complicated separation process is required. Furthermore, the above-mentioned method of producing L-phenylalanine which starts with an N-acyl-DL-phenylalanine is disadvantageous in that the salt of the N-acyl-DL-phenylalanine with a base tends to inhibit the enzyme activity in the reaction system to thereby cause marked decrease in enzyme activity. In the method of producing L-phenylalanine which uses a DL-phenylalanine alkyl ester as the starting material, the use of a lower alkyl ester of DL-phenylalanine is preferable because of quick formation of L-phenylalanine but disadvantageous in that the lower alkyl ester of DL-phenylalanine is easily converted to DL-phenylalanine by hydrolysis other than the hydrolysis under the action of esterase, so that it is difficult to attain a satisfactory high optical purity of L-phenylalanine using such starting material.
The method of producing L-phenylalanine which comprises treating 5-benzylhydantoin with hydantoinase has drawbacks that the starting material, namely 5-benzylhydantoin, is expensive and that the hydantoinase used is not satisfactorily active.
The method of producing L-phenylalanine which comprises treating an N-acyl-DL-phenylalanine methyl ester with serine proteinase or esterase, separating the resulting N-acyl-L-phenylalanine from the unreacted N-acyl-D-phenylalanine methyl ester and hydrolyzing the former with hydrochloric acid is disadvantageous in that the independent performance of hydrolysis of the ester residue and hydrolysis of the acid amide residue in the N-acylphenylalanine methyl ester makes the process troublesome.